Expression of Lyt differentiation antigens has been examined on murine thymocytes by flow cytometry, using fluorochrome - labeled monoclonal antibodies to the antigens. Two-color flow cytometric analysis revealed at least 4 populations of cells, one of which has not been described before. The size, morphology, cell cycle kinetics and surface phenotype suggest that this cell is immature and/or dividing; it is perhaps an early precursor of more mature thymocytes. Thymus obtained early after transplantation, and thymi from animals recovering from treatment with steroids show enrichment of this cell during repopulation. These cells bear Ly 9 antigen, proving that they are lymphoid. Analysis with several other monoclonal antibodies shows a phenotype not shared by any peripheral cell examined. Although this population constitutes only 3-4% of the thymocyte population, it is readily isolated free from contaminants by multiparameter (2 color, red and green, and size) cell sorting.